The present invention relates generally to the field of neurological and physiological dysfunctions associated with neuropsychiatric and developmental diseases, especially Alzheimer""s Disease. More particularly, the invention is concerned with the identification of proteins associated with neuropsychiatric and developmental diseases, especially Alzheimer""s Disease, and relates to methods of diagnosing these diseases, and to methods of screening for candidate compounds which modulate the interaction of a certain protein, specifically Presenilin Associated Membrane Protein (xe2x80x9cPAMPxe2x80x9d), with presenilin proteins.
Alzheimer""s Disease (AD) is a degenerative disorder of the human central nervous system characterized by progressive memory impairment and cognitive and intellectual decline during mid to late adult life (Katzman, N Eng J Med 1986;314:964-973). The disease is accompanied by a constellation of neuropathologic features principal amongst which are the presence of extracellular amyloid or senile plaques, and neurofibrillary tangles in neurons. The etiology of this disease is complex, although in some families it appears to be inherited as an autosomal dominant trait. Genetic studies have identified three genes associated with the development of AD, namely: (1) xcex2-amyloid precursor protein (xcex2APP) (Chartier-Harlin et al., Nature 1991;353:844-846; Goate et al., Nature 1991;349:704-706; Murrell et al., Science 1991:254:97-99; Karlinsky et al., Neurology 1992;42:1445-1453; Mullan et al., Nature Genetics 1992;1:345-347); (2) presenilin-1 (PS1) (Sherrington et al., Nature 1995;375:754-760); and (3) presenilin-2 (PS2) (Rogaev et al., Nature 1995;376:775-778; Levy-Lehad et al., Science 1995; 269:970-973).
Abnormal processing of xcex2APP with overproduction of amyloid-xcex2 is also a feature of other CNS diseases, including inherited and sporadic forms of amyloid angiopathy, which presents with intracerebral bleeding (Levy et al., Science 1990;248:1124-1126). Thus, abnormalities of presenilin proteins and PS-interacting proteins may affect these diseases as well.
The presenilin genes (PS1xe2x80x94PS1 and PS2xe2x80x94PS2) encode homologous polytopic transmembrane proteins that are expressed at low levels in intracellular membranes including the nuclear envelope, the endoplasmic reticulum the Golgi apparatus and some as yet uncharacterized intracytoplasmic vesicles in many different cell types including neuronal and non-neuronal cells (Sherrington et al., supra; Rogaev et al., supra, Levy-Lahad et al., supra; Doan et al., Neuron 1996;17:1023-1030; Walter et al., Molec. Medicine 1996;2:673-691; De Strooper et al., J. Biol. Chem. 1997;272:3590-3598; Lehmann et al., J.Biol.Chem. 1997;272:12047-12051; Li et al., Cell 1997;90:917-927). Structural studies predict that the presenilins contain between six and eight transmembrane (TM) domains organized such that the N-terminus, the C-terminus, and a large hydrophilic loop following the sixth TM domain are located in the cytoplasm or nucleoplasm, while the hydrophilic loop between TM1 and TM2 is located within the lumen of membranous intracellular organelles (Doan et al., 1996; De Strooper et al., 1997; et al., 1997).
Missense mutations in the PS1 and PS2 genes are associated with the inherited forms of early-onset AD (Sherrington et al., Nature 1995;375:754-760; Rogaev, et al., Nature 1995;376:775-778; Levy-Lahad et al, Science 1995;269:970-973). Several lines of evidence have also suggested roles in developmental, apoptotic signalling and in the regulation of proteolytic cleavage of the xcex2-amyloid precursor protein (xcex2APP) (Levitan et al., Nature 1995;377:351-354; Wong et al., Nature 1997;387:288-292; Shen et al., Cell 1997;89:629-639; Wolozin et al., Science 1996;274:1710-1713; De Strooper et al., Nature 1998;391:387-390). Nevertheless, it remains unclear just how these putative functions are mediated, or how they relate to the abnormal metabolism of the xcex2APP associated with PS1 and PS2 mutations (Martin et al., NeuroReport 995;7:217-220; Scheuner et al., Nature Med. 1996;2:864-870; Citron et al., Nature Med. 1997;3:67-72; Duff et al., Nature 1996;383:710-713; Borchelt et al., Neuron 1996;17:1005-1013).
P1 and PS2 interact specifically with at least two members of the armadillo family proteins; neuronal plakophilin-related armadillo protein (Paffenholtz et al., Differentiation 1997; 61: 293-304; Paffenholtz et al., Exp Cell Res 1999; 250: 452-464; Zhou et al., Neuroreport 1997; 8: 2085-2090) and xcex2-catenin, that are expressed in both embryonic and post-natal tissues. Moreover, the domains of PS1 and PS2 that interact with these proteins have been identified. Mutations in PS1 and PS2 affect the translocation of xcex2-catenin into the nucleus of both native cells and cells transfected with a mutant PS gene. These interactions and effects are described in detail in co-pending commonly assigned U.S. application Ser. No. 09/227,725, filed Jan. 8, 1999, the disclosure of which is incorporated herein by reference.
The identification and cloning of normal as well as mutant PS1 and PS2 genes and gene products are described in detail in co-pending commonly assigned U.S. application Ser. Nos. 08/431,048, filed Apr. 28, 1995; Ser. No. 08/496,841, filed Jun. 28, 1995; Ser. No. 08/509,359, filed Jul. 31, 1995; and Ser. No. 08/592,541, filed Jan. 26, 1996, the disclosures of which are incorporated herein by reference.
There is speculation that onset of AD may be associated with aberrant interactions between mutant presenilin proteins and normal forms of PS-interacting proteins, and these changes may increase or decrease interactions present with normal PS1, or cause interaction with a mutation-specific PS-interacting protein. Such aberrant interactions also may result from normal presenilins binding to mutant forms of the PS-interacting proteins. Therefore, mutations in the PS-interacting proteins may also be implicated in the development of AD.
While the identification of normal and mutant forms of PS proteins has greatly facilitated development of diagnostics and therapeutics, a need exists for new methods and reagents to more accurately and effectively diagnose and treat AD. In addition, further insights into PS proteins and their interaction with other components may lead to new diagnostic and treatment methods for other related CNS diseases.
Applicants have discovered that PS1 and PS2 interact specifically with a transmembrane protein, herein referred to as xe2x80x9cPresenilin Associated Membrane Proteinxe2x80x9d or xe2x80x9cPAMPxe2x80x9d, which is expressed in multiple tissues (e.g., brain, kidney, lung, etc.). The product of this gene is therefore implicated in the biochemical pathways affected in Alzheimer""s Disease, and may also have a role in other dementias, amyloid angiopathies, and developmental disorders such as spina bifida. This gene, therefore, presents a new therapeutic target for the treatment of Alzheimer""s Disease and other neurologic diseases. In addition, PAMP nucleic acids, proteins and peptides, antibodies to PAMP, cells transformed with PAMP nucleic acids, and transgenic animals altered with PAMP nucleic acids that possess various utilities, as described herein for the diagnosis, therapy and continued investigation of Alzheimer""s Disease and other neurodegenerative disorders. Furthermore, mutant PAMP nucleic acids, proteins, or peptides, cells transfected with vectors comprising mutant PAMP nucleic acids, transgenic animals expressing mutant PAMP or peptides thereof, and their use in studying Alzheimer""s Disease and other neurodegenerative disorders, or developing improved diagnostic or therapeutic methods for such disorders, are presented herein.
Thus, the invention provides isolated and purified presenilin associated membrane protein (PAMP), or a functional fragment thereof, as well as nucleic acids encoding a PAMP. Preferred nucleotide and amino acid sequences are provided herein. The invention further provides probes and primers for PAMP genes. Preferred embodiments include sequences of at least 10, 15 or 20 consecutive nucleotides selected from the disclosed sequences.
The invention also provides isolated and purified mutant PAMP, or a functional fragment thereof, as well as nucleic acids encoding a mutant PAMP, and probes and primers for PAMP genes. Preferred nucleotide and amino acid sequences are provided herein.
Using the nucleic acid and amino acid sequences disclosed herein, methods for identifying allelic variants or heterospecfic homologues of a human PAMP and gene are provided. The methods may be practiced using nucleic acid hybridization or amplification techniques, immunochemical techniques, or any other technique known in the art. The allelic variants may include other normal human alleles as well as mutant alleles of PAMP genes which may be causative of Alzheimer""s Disease or other CNS diseases. The heterospecific homologues may be from other mammalian species, such as mice, rats, dogs, cats or non-human primates, or may be from invertebrate species, such as Drosophila melanogaster or Caenorhabditis elegans. Thus, it is another object of the invention to provide nucleic acids that encode allelic or heterospecific variants of the disclosed sequences, as well as the allelic or heterospecific proteins encoded by them.
The invention also provides vectors, and particularly expression vectors (e.g., cos-Tet vector), which include any of the above-described nucleic acids. It is a further object of the invention to provide vectors in which normal or mutant PAMP nucleic acid sequences are operably joined to exogenous regulatory regions to produce altered patterns of expression, or to exogenous coding regions to produce fusion proteins. Conversely, it is another object to provide nucleic acids in which PAMP regulatory regions are operably joined to exogenous coding regions, including standard marker genes, to produce constructs in which the regulation of PAMP genes may be studied and used in assays or therapeutics.
The invention further provides host cells and transgenic animals transformed with any of the above-described nucleic acids of the invention. The host cells may be prokaryotic or eukaryotic cells and, in particular, may be gametes, zygotes, fetal cells, or stem cells useful in producing transgenic animal models. In one embodiment, the transgenic animal contains a transgene encoding a normal or mutant PAMP, which is expressed in neural cells such that expression can be detected, e.g., by detecting PAMP, mRNA, or protein, and more preferably by detecting a neuroprotective or a neurodegenerative phenotype. For example, the animal might manifest one or more symptoms of a neurodegenerative disease. The animal may be a vertebrate or an invertebrate. In a preferred embodiment, the transgenic animal is a mouse, which encodes a human PAMP. The transgenic animal may further comprise a second transgene encoding a normal or mutant PS1, PS2, or xcex2APP.
In another embodiment, the invention provides an animal containing a nucleic acid that expresses a PAMP or a mutant PAMP at a higher or lower level relative to expression level in a wild-type animal. The animal may be prepared by homologous recombination mediated targeting of endogenous PAMP nucleic acid. In a preferred embodiment, the animal is prepared by translocation of P-elements or chemical mutagenesis.
The invention also provides a reconstituted system for measuring PAMP activity, comprising PAMP, a mutant PAMP, or functional fragments thereof, and a PAMP substrate. The reconstituted system may be a whole cell. Preferably, the whole cell contains a first nucleic acid that expresses said PAMP and a second nucleic acid that expresses the substrate. Preferably, the substrate comprises PS1 protein, PS2 protein, xcex2APP, or a surrogate synthetic substrate protein such as Notch, which undergoes proteolytic processing events similar to those of xcex2APP (Haass C and Selkoe D J. Nature 1998; 391: 387-390; De Strooper B, et al., Nature 1999; 398:518-522; Song W, et al., Proc Natl Acad Sci USA 1999; 96: 6959-6963.; Struhl G and Greenwald I, Nature 1999; 398: 522-525; Ye Y, et al., Nature 1999; 398: 525-529.).
The invention provides, in addition, a complex between a PAMP, or a mutant PAMP, and an agent which provides a detectable conformational or functional change in the PAMP upon interaction with a substance being analyzed for activity st a neurodegenerative disease. The complex may further comprise PS1 protein, PS2 protein or xcex2APP.
The invention also provides a method for detecting a mutation in PAMP associated with Alzheimer""s or a related neurological disorder, comprising obtaining a nucleic acid sample from an individual diagnosed with or suspected of having a neurodegenerative disorder, and sequencing a gene encoding PAMP from said sample.
The invention also invention provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining a nucleic acid sample from an individual diagnosed with or suspected of having a neurodegenerative disorder, and sequencing a gene encoding PAMP from said sample.
The invention also provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining cells that contain nucleic acid encoding PAMP, and under non-pathological conditions, transcribe the nucleic acid, and measuring a level of transcriptional activity of the nucleic acid.
The invention further provides a method for diagnosing individuals predisposed to or having a neurodegenerative disorder, comprising obtaining cells from an individual that express nucleic acid encoding PAMP, or isolating PAMP from said individual, and measuring PAMP activity, for example PAMP expression levels. In an alternative embodiment, the activity or abundance of a PAMP substrate may be measured.
The invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising administering one or more putative agents to a transgenic animal and detecting a change in PAMP activity.
The invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising adding one or more said agents to the reconstituted system described above, and detecting a change in PAMP activity.
The invention also provides a method for identifying putative agents having anti-neurodegenerative activity, comprising adding one or more said agents to the complex described above, and detecting a conformational change in PAMP.
The invention also provides a method for identifying proteins that interact with PAMP, comprising contacting said substance to the reconstituted system discussed above, and detecting a change in PAMP activity.
Further the invention provides for a method for identifying substances that modulate PAMP activity, comprising contacting a sample containing one or more substances with PAMP, or a PAMP mutant, or functional fragments thereof, and a PAMP substrate, measuring PAMP activity, and determining whether a change in PAMP activity occurs. In a preferred embodiment, the substance is a PAMP inhibitor. In another preferred embodiment, the substance stimulates PAMP activity.
These and other aspects of the invention are further elaborated in the Detailed Description of the Invention and Examples, infra.